Near infrared (NIR) fluorescent dyes and biological probes using NIR labeled biomolecules or ligands have become important in biological systems due to their advantages over UV and visible fluorophores. As a result, the number of near-IR (NIR) fluorophores useful for biological systems has grown substantially in recent years. These long-wavelength fluorophores include for example, cyanine, oxazine, rhodamine, and phthalocyanine dyes.
Dyes that fluoresce in the NIR region can serve as a reporting group when tagged to biomolecules. One advantage of using NIR fluorescence technology is the almost complete elimination of background from autofluorescence of biomolecules. Another benefit to near-IR fluorescent technology is that the background from the scattered light from the excitation source is greatly reduced since the scattering intensity is proportional to the inverse fourth power of the wavelength. Low background fluorescence is necessary for highly sensitive detection. Furthermore, the optically transparent window in the near-IR region (650 nm to 900 nm) in biological tissue makes NIR fluorescence a valuable technology for in vivo imaging and subcellular detection applications that require the transmission of light through biological components.
Another attraction of NIR fluorescence for biological applications is the availability and low cost of long-wavelength diode lasers for excitation and high efficiency silicon avalanche photodiodes for detection. However, commercial diode lasers are only available at a limited number of discrete wavelengths. To achieve optimum excitation, a fluorophore's maximum absorption wavelength should match the laser wavelength.
Cyanine dyes have been widely used for labeling biomolecules, e.g. antibodies, DNA probes, avidin, streptavidin, lipids, biochemical analogs, peptide, drug for a variety of applications such as DNA sequencing, DNA microarray, western blotting, flow cytometry analysis, protein microarray. See, for example, U.S. Patent Application No. 20040014981 to Lugade, et al. published Jan. 22, 2004, and U.S. Pat. No. 5,268,486, incorporated herein by reference, for exemplary cyanine dyes. Scientists favor using cyanine dyes in biological applications because, among other reasons, cyanine dyes 1) are biocompatible; 2) have high molar absorptivity (c.a. 105 M−1 cm−1); 3) are readily modified to match a wide range of desired excitation and detection wavelengths (e.g. about 500 to about 900 nm); 4) are capable of incorporating water-soluble groups and linking groups; 5) and possess favorable fluorescence properties.
In certain applications, for example, detection of enzyme activity, screening of potential inhibitors in high throughput applications, detection of ligand-receptor interactions, and nucleic acid hybridizations, it is desirable to “quench” the fluorescence of the biomolecule or ligand. In order to monitor the biological process, the modulation of the fluorescence becomes important.
In one aspect, the modulation of fluorescence in a biomolecule can be achieved by, for example, labeling the biomolecule with a sufficient number of fluorescent dyes to an extent that the dyes are sufficiently quenched in the biomolecule, that the biomolecule is a substantially non-fluorescent substrate. Haughland, R. P. et al. (U.S. Pat. No. 5,719,031) describe polymers that are labeled with multiple borapolyaza-s-indacene fluorescent dyes (BODIPY) to the point such that fluorescence quenching occurs. Haughland R. P. et al. further describe that the degradation of the labeled polymer results in fluorescence enhancement and that the resulting fluorescence enhancement is useful for measuring the degradation of such polymers, for example, as a result of enzymatic hydrolysis of a protein, carbohydrate, nucleic acid, or other natural or synthetic polymer.
Assays for the detection of protease enzyme activity based on the self-quenching of fluorescence of visible fluorophores located on a small protein, e.g., casein or bovine serum albumin (BSA), have been described by various researchers. Commercial assay kits using this technology exist (Jones, L. J., et al., Analytical Biochemistry, 251, 144-152 (1997); Boonacker, E., et al., The Journal of Histochemistry & Cytochemistry, 49 (12), 1473-1486 (2001); Thompson, V. F., et al., Analytical Biochemistry, 279, 170-178 (2000); “One-step fluorescence-based protease assays,” Molecular Probes technical information). These commercial assays are generic protease assays that are designed to inform the assay user of the presence or absence of protease enzymes, and thus rely on the use of a “generic protease substrate,” meaning a substrate that is generally susceptible to proteolytic degradation by many proteases, as the protease substrate in the assay. Generic protease substrates can detect the presence of a variety of proteases and are can be especially important for assessing protease contamination in cells, cell lysates, tissue extracts, purified enzymes and purified recombinant proteins. Generic protease substrates can also be useful for comparing overall enzymatic activity in normal cells/tissues and diseased cells/tissues.
However, as the current protease assays available for detecting general protease activity all use “general protease substrates” having visible-range fluorophores associated therewith, these current assays suffer from the drawbacks of undesired background fluorescence (either from biological sample, library compounds, microplate, or scattering photons), and low assay sensitivity, all of which greatly hinder the utility of the current assays.
In another aspect, the modulation of fluorescence in a fluorescent probe can be achieved by associating a reporter-quencher dye pair with a biomolecule or ligand either through conjugation of each dye of the reporter-quencher dye pair to the same biomolecule, or through specific binding of a reporter-dye labeled biomolecule or ligand to a quencher-dye labeled biomolecule or ligand. Biological applications based on reporter-quencher dye pairs include, for example, kinase assays, nucleic acid hybridization assays, protein-protein interaction assays and protease assays. A protease assay of this type is typically configured such that a reporter-quencher dye pair is associated with a peptide protease substrate. Unlike a generic protease assay, described above, this type of protease assay is highly specific, and requires that certain peptide sequences in the protease substrate are recognized, bound and proteolytically processed by a specific protease. Protease assays designed to detect the activity of a specific protease are useful for detecting the presence of a specific protease in cells, cell lysates, tissues/tissue extracts; and are generally amenable to screening drug compound libraries for “hits” that is, compounds that are, for example, antagonists or agonists of the specific protease of interest, in which such modulation of protease activity may have therapeutic value.
Many organic dyes may be used as quenchers in bioassays as long as the spectrally matched fluorophore-quencher pair can be brought into proximity with proper alignment. However, many organic dyes that might be used as quenchers have intrinsic fluorescence. This can result in high background fluorescence and hence attenuate the sensitivity of assays. Dark quenchers with little or no intrinsic fluorescence can efficiently quench the fluorescence from the proximate fluorophores with little background when a large extent of spectral overlap exists between the emission spectrum of the donor/reporter fluorophore and the absorption spectrum of the acceptor quencher dye. Among the dark quenchers, an azo dye, 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL) is a widely used dark quencher in many assays, such as “molecular beacons” for DNA detection (see, U.S. Pat. No. 5,989,823) and protease assay for protease activity or inhibition detection (see, Science, 1990, 247(4945), 954-958). However, the absorption wavelength region (absorption maxima around 540 nm) for DABCYL quenchers restricts the utility of these compounds to short wavelength fluorophores donors. Diazo dyes of the BHQ series, which are referred to as “Black Hole Quenchers” (International Patent Publication No. WO 01/86001), provide a broad range of absorption with absorption maxima around 670 nm which overlaps well with the emission of many fluorophores in the visible region. The QSY series dyes from Molecular Probes are another series of dark quenchers having little or no observable fluorescence which absorbs maximally above 530 nm. The QSY dyes are derivatives of 3- and/or 6-amino xanthenes that are substituted at one or more amino nitrogen atoms by an aromatic or heteroaromatic quenching moiety and are used extensively as quenching reagents in many bioassays (see, U.S. Pat. No. 6,399,392). Non-fluorescent cyanine type dyes have also been developed. A class of nitro-substituted non-fluorescent asymmetric cyanine dye compounds (see, U.S. Pat. Nos. 6,750,024; 6,348,596; and 6,080,868) are useful in the context of a reporter-quencher energy transfer compound pair, particularly useful in nucleic acid hybridization assays employing fluorescence energy transfer as a means of detection. This class of quencher dyes also have short absorption wavelength thus limiting their pairings to short wavelength xanthene fluorescent donor dyes such as fluorescein and rhodamine dyes.
Another problem associated with the current dark quencher dyes is their poor water solubility due to their highly hydrophobic structure characteristic. Poor water solubility of the quencher dyes limits their applications in biological assays as their corresponding bioconjugates 1) are more difficult to prepare and purify; 2) are often not sufficiently soluble in the aqueous assay medium; 3) and exhibit undesired non-specific binding with other biomolecules in the assay.
In view of the foregoing, there remains a need for new NIR reporter dyes, NIR quencher dyes and biological probes having associated therewith NIR reporter and/or quencher dyes; and for biological assays using NIR dye technology that overcome the disadvantages of assays based on visible fluorophores. A need for NIR quenchers that have good water solubility, have large spectral overlap of their absorption spectra with most NIR fluorophores of interest, and are essentially non-fluorescent over a broad pH range also exists. Additionally, there remains a need for an improved generic protease assay technology. Surprisingly, the present invention satisfies these and other needs.